Project Summary/Abstract The incidence of multidrug resistant (MDR) bacterial infections is rising. This is particularly relevant for the Gram- positive bacterium Enterococcus faecalis, an opportunistic pathogen and leading cause of nosocomial bacteremia. The rise in MDR E. faecalis infections is largely attributed to their ability to resist many antibiotics including antibiotics of ?last resort?, such as vancomycin. E. faecalis is a native inhabitant of the human intestinal tract which serves as a major reservoir of MDR E. faecalis. Antibiotic depletion of the intestinal microbiota can facilitate blooms of E. faecalis leading to their enhanced dissemination to the bloodstream and other tissue sites. In many cases overgrowth of intestinal enterococci is dominated by vancomycin resistant enterococci (VRE) which severely limits available treatment options. With increasing VRE infections worldwide, it is imperative that new strategies for therapeutic intervention are explored. An alternative to traditional antibiotics is the use of biological agents for the treatment of drug resistant bacterial infections. One approach is harnessing bacteriophages (phages) that infect and kill bacteria. Although phages may hold promise as next generation therapeutics, we know little of the basic principles of phage infection processes in vivo and how the mammalian host environment influences phage-bacteria interactions. This project investigates the use of phages for the decolonization of VRE within its natural intestinal habitat. Using a combination of mouse intestinal colonization models, molecular genetics and whole genome sequencing, we aim to gain mechanistic insight into how intestinal selective pressures influence the infectivity and genome evolution of phages during phage-mediated VRE decolonization. To achieve this goal we will execute three specific aims: 1) Define the intestinal selective pressures that dictate phage-mediated E. faecalis decolonization, 2) Determine the long-term stability and genomic evolution of E. faecalis phages in the intestine, and 3) Determine cognate receptors for diverse E. faecalis phages and delineate receptor function. Understanding intestinal selective pressures and molecular mechanisms of enterococcal phage infection in vivo will aid in the development of novel phage therapeutics for the decolonization of enterococci recalcitrant to conventional antibiotics.